Transduction is the transfer of genetic material into cells via viral or phage particles.
Lentiviral vectors are important delivery systems especially due to their ability to sustain the expression over several months and to transduce both dividing and non-dividing cells. The system is based on the HIV virus, with modifications for safety. Only the viral proteins which are necessary for the production of the vectors are present in the 3 plasmids used to produce the vectors. The transfer vector (for example: pSEW and pSEW derived vectors), containing the gene to be brought into the cells, is the only vector which contains the packaging signal Ψ, meaning this is the only plasmid packaged into the viral particles. It also contains a SIN (self-inactivating) LTR, which means that there is a deletion in the 3’ LTR region, removing the LTR promoter activity and reducing the threat of recombination. The packaging construct (for example: pCMV∆R8.91) has the Gag-Pro-Pol, Tat, Rev, RRE proteins controlled under a CMV promoter. The third plasmid (for example: pCMV-G) contains the envelope protein, in this case from the VSV virus which enables the transduction of most cells.
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3.4.1 Lentiviral vector production
Production of the lentiviral vectors was achieved with calcium phosphate transfection of the three plasmids (pSEW or pSEW derived vectors, pCMV∆R8.91, and pCMV-G) into HEK-293T cells. Calcium phosphate transfection brings the DNA into the cells via precipitates of the plasmid DNA formed by its interaction with the calcium ions which are taken up by the cell through endocytosis. The HEK-293T cells are plated the day before in 10 cm culture dishes with 6x106 cells per dish. On the day of the transfection, 25 mM Chloroquine is added to the cells at a dilution of 1:1000. The DNA is then mixed together as follows: 7.5 µg transfer vector (the pSEW derived plasmids), 12.5 µg packaging construct (pCMV∆R8.91), and 2 µg of the envelope plasmid (pCMV-G) and filled to 450 µl with sterile water (an additional plasmid, a eukaryotic vector containing the Vif protein (1 µg) was also added to the packaging of the LLVs which contain the gene for APOBEC3G). Then 50 µl of 2.5 mM CaCl2 was added to the DNA mixture. The DNA/CACl2 solution was then added dropwise to 500 µl 2x HEPES buffer while vortexing. The mixture was incubated for 20 min at room temperature and then 1 ml was added to each plate. The cells were incubated under normal cell culture conditions for 6-8 hrs after which time the medium was changed. The supernatants were collected 24, 36 and 48 hrs after transfection and directly filtered with a syringe top sterile filter (0.22µm). The filtered supernatant was pooled and kept at 4˚C until being concentrated and frozen at -80ºC. (see appendix for the plasmid maps)
3.4.2 Concentrating lentiviral vectors
Lentiviral vectors were concentrated using the ultracentrifuge. For this, 34 ml of the culture supernatant was filled into each tube. This was then centrifuged for 2 hrs at 19500xg and the supernatant was discarded. The pellet was resuspended overnight at 4˚C in the medium left in the tube (approximately 500 µl) and was then aliquoted, frozen and titrated.
3.4.3 Titration of lentiviral vectors
To determine the amount of lentiviral particles are in a milliliter, the viral stock was titrated.
For the titration, 5x104 cells per well were seeded in a 24 well plate one day before titrating the viruses. A dilution series of the lentiviral vectors was done, in the case of the concentrated vectors they were diluted as follows: 1:100,000; 1:10,000, 1:5000; 1:1000;
1:500; 1:100. The unconcentrated lentiviral vectors were diluted 1:10,000; 1:1000; 1:500;
1:100; 1:500; 1:100 in medium. The plates were centrifuged for 1 hr at 2000 rpm at 31ºC and incubated with standard cell conditions. After 48 hrs the cells could be analyzed by FACS and the titer calculated. The titer was calculated with cultures which had less than 20%
positive cells (to reduce the double positives) as follows (see figure13 for an example):
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0 1000
FSC-H
0 1000
FSC-H A3G_Vif_0_01.018 A3G_Vif_0_01.018
R2
Figure 13. FACS analysis of a titration with LVVs. For this analysis, using a dilution of 1:100,000 13.8% of the cells were eGFP positive, meaning they were transduced with the LVV.
Putting this into the formula it is: Titer = 13.8%/100 x 50,000 cells seeded x 100,000. Using this formula, the titer is 6.9x108 infectious particles /ml.
With the titer of the virus stock, the amount of virus needed for transducing cells can be determined. The MOI, also known as multiplicity of infection, is defined as being the amount of infectious particles per cell. An MOI of 1 is the same as having one virus per cell. The MOI needed to infect cells to the extent that on average only one virus enters each cell is different for each cell type. The MOI can be determined with the following formula:
MOI [infectious particles/cell] = titer [infectious particles/ml] x volume / cell number
The optimal MOI for certain cells can be determined by plotting the percent of positive cells against the MOI. In this case 50-100% positive HUT78 cells were required therefore the optimal MOI was approximately 5. This is shown in Figure14.
0 20 40 60
0%
20%
40%
60%
80%
100%
0.2763 +
) 0.1851Ln(x
= y
0.9841
= R2
MOI
e G F P P o s it iv e C e ll s [ % ]
Figure 14. Determining the optimal MOI to transduce cell lines. In this case LVVs titered on Te671 cells were tested to determine the optimal MOI to use in HUT78 cells.
13.8%
Titer [infectious particles/ml] = (positive transduced cells (%)/100) x seeded cell
number x dilution factor
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3.4.4 Transduction
The cells are transduced by quickly thawing the concentrated or unconcentrated LVVs and adding the correct MOI to the cells. The cells are then centrifuged for 1hr at 2000 RPM and incubated over night in cell culture conditions. After at least 12 hrs, the medium is changed.
The transduction efficiency can be analyzed after 48 hrs by means of FACS (see section 3.2).